Journal: Life Science Alliance

Article Title: RAS and PP2A activities converge on epigenetic gene regulation

doi: 10.26508/lsa.202301928

Figure Lengend Snippet: (A) Cell viability inhibitory effect of increasing concentrations of panobinostat (HDACi) in PP2A-A–depleted cells as compared to siCtrl (n = 3). (B, C) Synergy between panobinostat and the PP2A activator DBK1154 as a dose–response matrix and with Bliss synergy area, respectively (n = 2). (D) Western blots showing the total protein levels of HDAC1/2 in whole-cell lysates of cells after RAS or PP2A manipulation. Left panel: siRNA-mediated inhibition of PP2A inhibitory proteins and RAS for 72 h in H460 cells; middle panel: pharmacological PP2A activation by drug treatment (10 µM, 48 h); and right panel: HBEC step-wise–transformed by shTP53 (p53−), mutant KRAS expression (KRAS+), and small-t–mediated PP2A inhibition (ST+). (E) Validation of the suitability of the reporter system for detecting gene activation upon pharmacological targeting of PP2A/RAS-regulated epigenetic mechanisms. Shown are relative amounts of GFP-positive HCT116 SFRP1-GFP reporter cells by IncuCyte analysis after 48 h of treatment with indicated drugs at the following IC50 cell viability concentrations (see ): azacitidine, 22.24 μM; decitabine, 10 μM; iBET151, 18.78 μM; JQ1, 5.26 μM; mivebresib, 3.09 μM; panobinostat, 0.278 μM; and TSA, 0.246 μM. (n = 2). (F) Western blot analysis of protein levels of WT and indicated mutants of FLAG-tagged RNF168 48 h after transient transfection. The samples are taken from parallel cells as used for reporter assays shown in .

Article Snippet: A549, H358, H460, and HBEC were authenticated (STR profiling) by the European Collection of Authenticated Cell Cultures in December 2018.

Techniques: Western Blot, Inhibition, Activation Assay, Transformation Assay, Mutagenesis, Expressing, Biomarker Discovery, Transfection

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Analysis of C15 infectivity by immunofluorescent staining. HBEC-ALI cultures were infected for 18 h with C15 and imaged apically and z-stacked orthogonally by confocal microscopy. Secretory and ciliated cells were stained with WGA and rabbit polyclonal antibody against aat, respectively ( false color red ), C15 capsid stained with monoclonal antibody ( false color green ), nuclei stained with Syto-13 ( false color blue ). Scale bars indicate 20 μm

Article Snippet: Preliminary experiments revealed that cells cultured using a different HBEC culture medium (PneumaCultTM, Stem Cell Technologies, Vancouver, Canada) had more plentiful ciliated cells [ ].

Techniques: Infection, Staining, Confocal Microscopy

Journal: Respiratory Research

Article Title: Rhinovirus C targets ciliated airway epithelial cells

doi: 10.1186/s12931-017-0567-0

Figure Lengend Snippet: Single cell analysis of RV-C infection and epithelial cell surface markers. HBEC-ALI cultures infected or mock-infected for 18 h with C15 or BEGM alone, respectively, were labeled with antibodies against C15 capsid (C15), and either a acetylated-alpha-tubulin (aat, Cilia) or b fluorescently labeled-WGA (WGA) and analyzed by flow cytometry. The graphs summarize the percentage of C15+ cells in ciliated and non-ciliated cells ( n = 4 independent experiments, two cell donors). Mock, mock-inoculated cultures; C15, C15-inoculated cultures. *** p < 0.001

Article Snippet: Preliminary experiments revealed that cells cultured using a different HBEC culture medium (PneumaCultTM, Stem Cell Technologies, Vancouver, Canada) had more plentiful ciliated cells [ ].

Techniques: Single-cell Analysis, Infection, Labeling, Flow Cytometry

Journal: PLoS ONE

Article Title: Monomethylarsonous Acid (MMA III ) Has an Adverse Effect on the Innate Immune Response of Human Bronchial Epithelial Cells to Pseudomonas aeruginosa

doi: 10.1371/journal.pone.0142392

Figure Lengend Snippet: LDH release by HBEC was used to assess arsenic cytotoxicity and was measured using the Promega CytoTox 96 Non-Radioactive Cytotoxicity assay per manufacturers instructions. Data reported as optical density (OD, at 490 nm) of the cell culture medium bathing 500K cells. The first two bars represent LDH released by cells lysed by Triton-X (Lysed Cells). Data presented as mean ± SEM. LDH release was not significantly different from 0 in vehicle and arsenic treated cells, with and without P . aeruginosa exposure. n = 4 donors per treatment group.

Article Snippet: Lactate dehydrogenase (LDH) release by HBEC was used to assess cytotoxicity of all treatments and was measured using the Promega CytoTox 96 Non-Radioactive Cytotoxicity assay per manufacturers instructions (Madison, WI).

Techniques: Cytotoxicity Assay, Cell Culture